Silencing of hERG1 Gene Inhibits Proliferation and Invasion, and Induces Apoptosis in Human Osteosarcoma Cells by Targeting the NF-κB Pathway.

Zeng W, Liu Q, Chen Z, Wu X, Zhong Y, Wu J - J Cancer (2016)

Bottom Line:
Knockdown of hERG1 significantly suppressed cellular proliferation and invasion, and induced apoptosis, while inhibition of hERG1 significantly decreased activation of NF-κB.Overall, hERG1 may stimulate nuclear translocation of p65, thus regulating the NF-κB pathway through the activation of the hERG1/beta1 integrin complex and PI3K/AKT signaling.Furthermore, this regulation by hERG1 is, at least in part, through mediation of the NF-κB pathway.

ABSTRACTRecently, the human ether à go-go (eag) related gene 1 (hERG1) channel, a member of the voltage-dependent potassium channel (Kv) family, was determined to have a critical role in cancer cell proliferation, invasion, tumorigenesis and apoptosis. However, the expression levels and functions of hERG1 in osteosarcoma cells remain poorly characterized. In this study, hERG1 transcript and protein levels in osteosarcoma cells and tissues were measured using semi-quantitative real time PCR (RT-PCR), Western blot, and immunohistochemistry. The effects of hERG1 knockdown on osteosarcoma cell proliferation, apoptosis and invasion were examined using CCK-8, colony formation, flow cytometry, caspase-3 activity, wound healing and transwell based assays. Furthermore, semi-quantitative RT-PCR, Western blot and a luciferase reporter assay were used to assess the effects of hERG1 inhibition on the nuclear factor-κB (NF-κB) pathway. In addition, the effect of NF-κB p65-siRNA and NF-κB p65 expression on the survival of osteosarcoma cells was investigated. Through this work, a relationship for hERG1 with the NF-κB pathway was identified. Osteosarcoma cells and tissues were found to express high levels of hERG1. Knockdown of hERG1 significantly suppressed cellular proliferation and invasion, and induced apoptosis, while inhibition of hERG1 significantly decreased activation of NF-κB. Overall, hERG1 may stimulate nuclear translocation of p65, thus regulating the NF-κB pathway through the activation of the hERG1/beta1 integrin complex and PI3K/AKT signaling. Taken together, these results demonstrate that hERG1 is necessary for regulation of osteosarcoma cellular proliferation, apoptosis and migration. Furthermore, this regulation by hERG1 is, at least in part, through mediation of the NF-κB pathway.

Mentions:
As shown in Fig. 2-4, hERG1 downregulation and/or inhibition prevented proliferation and invasion, and induced apoptosis of osteosarcoma cells. In order to detail the mechanism behind these phenotypes, we first focused on the NF-κB pathway. This pathway regulates the expression of a number of genes proposed to govern tumor survival, growth, apoptosis, invasion and angiogenesis 18-20. As shown in Fig. 5A, transfection of cells with hERG1-siRNA resulted in the downregulation of NF-κB-regulated gene products, including cIAP-1, Bcl2, Survivin, XIAP, and MMP-2 and MMP-9. Western blot confirmed these results (Fig. 5B). Furthermore, a decrease in IκBα phosphorylation and an increase in total IκBα protein levels in osteosarcoma cells occured following hERG1-siRNA transfection (Fig. 5B). Likewise, translocation of NF-κB p65 protein into the nucleus, an indicator of NF-κB transcriptional activity, and NF-κB-luciferase reporter activity were also lower in osteosarcoma cells transfected with hERG1-siRNA than control cells (Fig. 5B-C). Moreover, overexpression of hERG1 in MG-63 cells also led to increased nuclear localization of NF-κB p65 (Fig. 5D).

Mentions:
As shown in Fig. 2-4, hERG1 downregulation and/or inhibition prevented proliferation and invasion, and induced apoptosis of osteosarcoma cells. In order to detail the mechanism behind these phenotypes, we first focused on the NF-κB pathway. This pathway regulates the expression of a number of genes proposed to govern tumor survival, growth, apoptosis, invasion and angiogenesis 18-20. As shown in Fig. 5A, transfection of cells with hERG1-siRNA resulted in the downregulation of NF-κB-regulated gene products, including cIAP-1, Bcl2, Survivin, XIAP, and MMP-2 and MMP-9. Western blot confirmed these results (Fig. 5B). Furthermore, a decrease in IκBα phosphorylation and an increase in total IκBα protein levels in osteosarcoma cells occured following hERG1-siRNA transfection (Fig. 5B). Likewise, translocation of NF-κB p65 protein into the nucleus, an indicator of NF-κB transcriptional activity, and NF-κB-luciferase reporter activity were also lower in osteosarcoma cells transfected with hERG1-siRNA than control cells (Fig. 5B-C). Moreover, overexpression of hERG1 in MG-63 cells also led to increased nuclear localization of NF-κB p65 (Fig. 5D).

Bottom Line:
Knockdown of hERG1 significantly suppressed cellular proliferation and invasion, and induced apoptosis, while inhibition of hERG1 significantly decreased activation of NF-κB.Overall, hERG1 may stimulate nuclear translocation of p65, thus regulating the NF-κB pathway through the activation of the hERG1/beta1 integrin complex and PI3K/AKT signaling.Furthermore, this regulation by hERG1 is, at least in part, through mediation of the NF-κB pathway.

ABSTRACTRecently, the human ether à go-go (eag) related gene 1 (hERG1) channel, a member of the voltage-dependent potassium channel (Kv) family, was determined to have a critical role in cancer cell proliferation, invasion, tumorigenesis and apoptosis. However, the expression levels and functions of hERG1 in osteosarcoma cells remain poorly characterized. In this study, hERG1 transcript and protein levels in osteosarcoma cells and tissues were measured using semi-quantitative real time PCR (RT-PCR), Western blot, and immunohistochemistry. The effects of hERG1 knockdown on osteosarcoma cell proliferation, apoptosis and invasion were examined using CCK-8, colony formation, flow cytometry, caspase-3 activity, wound healing and transwell based assays. Furthermore, semi-quantitative RT-PCR, Western blot and a luciferase reporter assay were used to assess the effects of hERG1 inhibition on the nuclear factor-κB (NF-κB) pathway. In addition, the effect of NF-κB p65-siRNA and NF-κB p65 expression on the survival of osteosarcoma cells was investigated. Through this work, a relationship for hERG1 with the NF-κB pathway was identified. Osteosarcoma cells and tissues were found to express high levels of hERG1. Knockdown of hERG1 significantly suppressed cellular proliferation and invasion, and induced apoptosis, while inhibition of hERG1 significantly decreased activation of NF-κB. Overall, hERG1 may stimulate nuclear translocation of p65, thus regulating the NF-κB pathway through the activation of the hERG1/beta1 integrin complex and PI3K/AKT signaling. Taken together, these results demonstrate that hERG1 is necessary for regulation of osteosarcoma cellular proliferation, apoptosis and migration. Furthermore, this regulation by hERG1 is, at least in part, through mediation of the NF-κB pathway.